Within the past two decades, hospitals have been combating a major crisis: a rise in antibiotic resistant bacteria. To add to this crisis, there has been little success in the development of new therapeutics to treat these infections. The drug daptomycin, however, was recently approved to treat antibiotic-resistant Gram-positive bacteria. Although only used in hospital settings, resistant bacteria were discovered within two years after daptomycin was approved for use. Oftentimes, bacteria become resistant to antibiotics by genetic exchange of drug resistance cassettes; however, these daptomycin resistant bacteria did not acquire new genes but instead had mutations in genes involved in cell membrane formation or modification. Thus, membrane alterations contribute to resistance. The bacterial pathogen Enterococcus faecalis, a major cause of hospital-acquired infections, can incorporate fatty acids from host fluids which in turn leads to increased daptomycin resistance. This is not due to genetic mutation but rather physiological changes that occur upon growth in fatty acid sources. Furthermore, this increased daptomycin resistance is not due to activation of previously described pathways. This proposal is designed to address how altered membrane compositions leads to decreased sensitivity of E. faecalis to daptomycin. Specifically, it will examine how different fatty acids impact daptomycin resistance and how altered membrane composition influences interactions with the antibiotic (Aim 1, 3). This proposal will also evaluat the roles of newly identified proteins required for fatty acid incorporation and how these proteins contribute to alterations in daptomycin sensitivity. As growth in fatty acid sources leads to increased levels of the phospholipid cardiolipin, experiments will be performed discern which factor, fatty acid composition, or phospholipid content, contributes the most to decreased sensitivity to daptomycin (Aims 2, 3). This analysis will be combined with whole transcriptomic and metabolomic profiling on wild type and specific genetic mutant strains cultured in the presence and absence of fatty acids and upon exposure to daptomycin (Aim 3). Together, these studies will elucidate how fatty acid and phospholipid alterations can impact sensitivity to daptomycin and the potential use of fatty acid transport inhibitors for antibiotic development.